This invention relates generally to the field of confocal microscopes and two-photon fluorescence microscopy. In particular, it is related to an assembly of fiber-coupled, angled-dual-illumination-axis confocal scanning microscopes with integrated structure, enhanced resolution, higher sensitivity, and versatile imaging capabilities.
The advent of fiber optics and laser technology has brought a renewed life to many areas of conventional optics. Confocal microscopes, for example, have enjoyed higher resolution, more integrated structure, and enhanced imaging capability. Consequently, confocal microscopes have become increasingly powerful tools in a variety of applications, including biological and medical imaging, optical data storage and industrial applications.
In recent years, a great deal of ingenuity has accordingly been devoted to improving the axial resolution of confocal microscopes using high numerical aperture (NA) lenses. A particularly effective approach is to spatially arrange two separate illumination and observation objective lenses, or illumination and observation beam paths, in such a way that the illumination beam and the observation beam intersect at an angle theta (xcex8) at the focal points, so that the overall point-spread function for the microscope, i.e., the overlapping volume of the illumination and observation point-spread functions results in a substantial reduction in the axial direction. A confocal microscope with such an angled, dual-axis design is termed a confocal theta microscope, or an angled-dual-axis confocal microscope, hereinafter. Its underlying theory is described below for the purpose of elucidating the principle of the present invention. A more detailed theory of the confocal theta microscopy can be found in xe2x80x9cFundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopyxe2x80x9d of Stelzer et al., Optics Communications 111 (1994), pp.536-547; U.S. Pat. No. 5,973,828; xe2x80x9cConfocal microscope with large field and working distancexe2x80x9d of Webb et al., Applied Optics, Vol.38, No.22, pp.4870; and xe2x80x9cA new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopyxe2x80x9d of Stelzer et al., Journal of Microscopy, Vol.179, Part 1, pp. 1; all incorporated by reference. It should be noted that high NA objectives are used in these prior art systems to achieve high resolution.
The region of the point-spread function of a confocal microscope""s objective that is of most interest is the region in which the point-spread function reaches its maximum value. This region is referred to as the xe2x80x9cmain lobexe2x80x9d of the point-spread function in the art. It is typically characterized in three dimensions by an ellipsoid, which extends considerably further in the axial direction than in the transverse direction. This characteristic shape is the reason that the axial resolution is inherently poorer than the transverse resolution in a conventional confocal microscope. When the main lobes of the illumination and observation point-spread functions are arranged to intersect at an angle in a confocal theta microscope, however, a predominantly transverse and therefore narrow section from one main lobe is made to multiply (i.e., zero out) a predominantly axial and therefore long section from the other main lobe. This optimal multiplication of the two point-spread functions reduces the length of the axial section of the overall point-spread function, thereby enhancing the overall axial resolution. The shape of the overall point-spread function can be further adjusted by varying the angle at which the main lobes of the illumination and observation point-spread functions intersect.
In addition to achieving higher resolution, an angled-dual-axis confocal microscope described above renders a number of additional important advantages. For instance, since the observation beam is positioned at an angle relative to the illumination beam, scattered light along the illumination beam does not easily get passed into the observation beam, except in the region where the beams overlap. This substantially reduces scattered photon noise in the observation beam, thus enhancing the sensitivity and dynamic range of detection. Moreover, by using low NA focusing elements (or lenses) for focusing the illumination and observation beams, the illumination and observation beams do not become overlapping until very close to the focus. Therefore, such an arrangement prevents scattered light in the illumination beam from directly xe2x80x9cjumpingxe2x80x9d to the corresponding observation beam, thereby further filtering out scattered photon noise in the observation beam. As such, an angled-dual-axis confocal microscope using relatively low NA lenses has much lower noise and is capable of providing much higher contrast when imaging in a scattering medium, rendering it highly suitable for imaging within biological specimens.
The aforementioned angled-dual-axis confocal arrangement can be further utilized to perform two-photon (and multi-photon) fluorescence microscopy, so as to exploit its high resolution and low noise capabilities. In such an arrangement, two illumination beams are directed to intersect optimally, such that each of the two observation beams thus produced is in an optimal confocal arrangement with its corresponding illumination beam.
Whereas traditional single-photon fluorescence laser microscopy requires only a single photon xcex3 for excitation, two-photon fluorescence microscopy requires simultaneous absorption of two photons xcex1 and xcex2 for excitation. In terms of energy, hc/xcex3 =hc/xcex1+hc/xcex2. Thus, xcex1 and xcex2 are both longer in wavelength than xcex3. However, it is important to note that xcex2 need not necessarily equal xcex1. Indeed, any combination of wavelengths can be used, so long as the net energy requirements for exciting the particular types of fluorophores being used are satisfied. An inherent advantage of two-photon fluorescence is that the two-photon absorption occurs only within a confined region where the two incident beams overlap, hence eliminating unwanted, spurious fluorescence and scattered light. Moreover, because two-photon excitation depends on the square of the excitation power, the excited volume is restricted to the focal point, providing an equivalent of confocal conditions. Additional advantages provided by two-photon (and multi-photon) excitation include longer penetration depth within a specimen (since longer wavelengths are employed, thus reducing scattering losses), reduced photobleaching and phototoxicity, and reduced background noise.
Accordingly, two-photon excitation has been of considerable interest for microscopy, fluorescence spectroscopy, and for single-molecule detection. For instance, two-photon fluorescence microscopy has been used in the art for imaging various types of fluorophores (or fluorophore indicators attached to proteins and biological cells) that are of particular interest to biomedical applications. It has also been used as an alternative way of attaining enhanced resolution and greater flexibility in imaging. The prior art effort in utilizing two-photon microscopy is exemplified by U.S. Pat. No. 5,034,613 of Denk et al.; U.S. Pat. No. 6,020,591 of Harter et al.; xe2x80x9cTwo-color Two-Photon Excitation of Fluorescencexe2x80x9d by Lakowicz et al. in Photochemistry and Photobiology, 64(4), (1996) pp.632-635; xe2x80x9cCombined scanning optical coherence and two-photon-excited fluorescence microscopyxe2x80x9d by Beaurepaire et al. in Optics Letters, Vol.24, No.14, (1999) pp. 969-971; and xe2x80x9cResolution improvement in nonconfocal theta microscopyxe2x80x9d by Lindek et al. in Optics Letters, Vol.24, No.21, (1999) pp.1505-1507. None of these prior art systems, however, exploit advantages gained by using relatively low NA lenses and hence allow themselves to be miniaturized or have sufficiently long working distances needed for in-vivo biological applications. Moreover, the scanning mechanisms employed in some of these systems are designed such that they do not lend themselves to fast speed and high precision scanning.
Furthermore, in recent years optical fibers have been used in confocal systems to transmit light more flexibly. A single-mode fiber is typically used so that an end of the fiber is also conveniently utilized as a pinhole for both light emission and detection. This arrangement is not susceptible to the alignment problems the mechanical pinholes in the prior art systems tend to suffer. Moreover, the use of optical fibers enables the microscopes to be more flexible and compact in structure, along with greater maneuverability in scanning.
Hence, what is needed in the art is a confocal scanning microscope for performing single-photon, two-photon, and higher order multi-photon fluorescence microscopy that achieves enhanced axial resolution, higher sensitivity and larger dynamic range of detection, a longer working distance and a larger field of view, fast and high precision scanning. The desired confocal scanning microscope should also have an integrated and scalable structure, rendering it a modular and versatile device, and further capable of being miniaturized for in-vivo biological applications.
Accordingly it is a principal object of the present invention to provide an angled-dual-illumination-axis confocal scanning microscope that utilizes two illumination beams in an angled-dual-axis confocal arrangement, so as to exploit enhanced axial resolution and higher sensitivity rendered by the angled-dual-axis confocal arrangement. The angled-dual-illumination-axis confocal scanning microscope provides additional advantages of high-speed scanning, larger dynamic range of detection, a larger field of view, a longer working distance, and a compact and integrated construction. Moreover, the angled-dual-illumination-axis confocal scanning microscope of the present invention is highly versatile in its imaging capabilities, providing an assortment of reflectance images and fluorescence images of many types, including single-photon fluorescence, two-photon fluorescence, and higher order multi-photon fluorescence.
It is another further object of the present invention to provide an assembly of fiber-based angled-dual-illumination-axis confocal scanning systems that advantageously combine the angled-dual-illumination-axis confocal scanning microscope of the present invention and fiber-optic components.
It is a further object of the present invention to provide a miniaturized angled-dual-illumination-axis confocal scanning microscope for in-vivo biological applications.
These and other objects and advantages will become apparent from the following description and accompanying drawings.
This invention provides an angled-dual-illumination-axis confocal scanning microscope, comprising an angled-dual-illumination-axis confocal scanning head and a vertical scanning unit. The angled-dual-illumination-axis confocal scanning head further comprises first and second optical fibers, an angled-dual-illumination-axis focusing means, and a scanning means.
From a first end of the first optical fiber a first illumination beam with a first wavelength emerges. The angled-dual-illumination-axis focusing means serves to focus the first illumination beam to a first diffraction-limited focal volume along a first axis within an object. From a first end of the second optical fiber a second illumination beam with a second wavelength emerges. The angled-dual-illumination-axis focusing means focuses the second illumination beam to a second diffraction-limited focal volume along a second axis within an object. The angled-dual-illumination-axis focusing means further receives a first observation beam emanated from the second focal volume within the object, and focuses the first observation beam to the first end of the second optical fiber. The angled-dual-illumination-axis focusing means additionally receives a second observation beam emanated from the first focal volume within the object, and focuses the second observation beam to the first end of the first optical fiber. The angled-dual-illumination-axis focusing means is so designed that the first and second axes intersect at an angle xcex8 within the object, such that the first and second focal volumes intersect optimally at a confocal overlapping volume. The scanning means, in the form of a single scanning element disposed between the angled-dual-illumination-axis focusing means and the object, is positioned such that it receives the first and second illumination beams from the angled-dual-illumination-axis focusing means and directs the illumination beams to the object. The scanning means further collects the first and second observation beams emanated from the object and passes the observation beams to the angled-dual-illumination-axis focusing means. Moreover, the scanning means is capable of pivoting the illumination beams and their corresponding observation beams jointly in such a way that the first and second axes remain intersecting optimally at a fixed angle xcex8 and that the confocal overlapping volume moves along an arc-line within the object, thereby producing an arc-line scan.
The vertical scanning unit comprises a vertical translation means and a compensation means. The vertical translation means is mechanically coupled to the angled-dual-illumination-axis confocal scanning head, such that it causes the angled-dual-illumination-axis confocal scanning head to move towards or away from the object, whereby a succession of arc-line scans that progressively deepen into the object is produced, providing a two-dimensional vertical cross-section scan of the object. The compensation means keeps the optical path lengths of the illumination and observation beams substantially unchanged, thereby ensuring the optimal intersection of the first and second focal volumes in the course of vertical scanning. Altogether, the angled-dual-illumination-axis confocal scanning microscope of the present invention is designed such that it is capable of performing vertical cross-section scanning in a line-by-line fashion with enhanced axial (i.e., vertical) resolution and greater speed, while maintaining a workable working distance and a large field of view. Additionally, the object may be moved incrementally in a direction perpendicular to the vertical cross-section scan plane as each vertical cross-section scan is completed, resulting in a plurality of vertical cross-section images that can be assembled into a three-dimensional image of a region within the object.
It should be understood that when describing the intersection of the illumination and observation beams in this specification, the term xe2x80x9coptimalxe2x80x9d means that the first and second focal volumes [i.e., the main lobe of the first illumination beam""s point-spread function (which coincides with the main lobe of the second observation beam""s point-spread function) and the main lobe of the second illumination beam""s point-spread function (which coincides with the main lobe of the first observation beam""s point-spread function)] intersect in such a way that their respective centers substantially coincide and the resulting overlapping volume has comparable transverse and axial extents. This optimal overlapping volume is termed xe2x80x9cconfocal overlapping volumexe2x80x9d in this specification. As such, in the angled-dual-illumination-axis arrangement described above, the first and second illumination beams intersect optimally with their corresponding observation beams respectively. It should be noted that in this arrangement, the axis of the first observation beam is collinear with the axis of the second illumination beam, however with the optical power of each beam traveling in the opposite directions. Likewise, the axis of the second observation beam is collinear with the axis of the first illumination beam, however with the optical power of each beam traveling in the opposite directions.
Moreover, the observation beams described above should be construed in a broad sense as each carrying any light transmitted back from the object, including reflected light, scattered light, and fluorescent light. The first and second illumination beams may have the same wavelength, for instance, in the infrared range. The fluorescence light thus produced would include one-color two-photon (and multi-photon) fluorescence. The first and second illumination beams may also have very different wavelengths. For instance, the first wavelength may be in the infrared range, while the second wavelength lies in the visible range. The fluorescence light thus obtained would include two-color two-photon (and possibly multi-photon) fluorescence. A skilled artisan will know how to selectively make use of a particularly type of light collected from the object and filter out spurious background light for a given application.
In addition to collecting first and second observation beams, the angled-dual-illumination-axis confocal scanning microscope of the present invention is equipped to provide one or more revenues for collecting additional light beams emanated from the object. As a way of example, a third observation beam comprising predominantly fluorescence light can be collected by the scanning means, and pivoted concurrently along with the first and second illumination beams and the corresponding first and second observation beams. The third observation beam thus collected is then directed to an auxiliary focusing means, which in turn focuses the third observation beam to an input end of a third optical fiber. In this case, the point-spread function of the third observation beam is caused to overlap with the confocal overlapping volume of the first and second illumination beams, hence yielding a higher resolution by way of an effective multiplication of the point-spread functions of all three beams. Accordingly, such a way of collection is termed xe2x80x9cconfocal-collectionxe2x80x9d, hereinafter. And the first, second and third optical fibers are preferably single-mode fibers in this case. Alternatively, the third observation beam can be collected by an auxiliary light collecting means positioned anywhere in the proximity of the objectxe2x80x94effectively xe2x80x9cstaringxe2x80x9d at the object, and is not in optical communication with the scanning means. The auxiliary light collecting means in turn focuses the third observation beam to an input end of a third optical fiber (or directs the third observation beam onto a xe2x80x9cstaringxe2x80x9d optical detector). The third fiber in this case is used for collecting light only, and is preferably a larger diameter multi-mode fiber, so as to maximize the collection efficiency of light emanating from the confocal overlapping volume throughout its motion within the object during scanning. Additionally, a light detector may be optically coupled to an output end of the third fiber, to detect light in the third observation beam. This way of collection is termed xe2x80x9cnon-confocal-collectionxe2x80x9d, hereinafter.
In an angled-dual-illumination-axis confocal scanning head of the present invention, the angled-dual-illumination-axis focusing means generally comprises an assembly of beam focusing, collimating, and deflecting elements. Such elements can be selected from the group of refractive lenses, diffractive lenses, GRIN lenses, focusing gratings, micro-lenses, holographic optical elements, binary lenses, curved mirrors, flat mirrors, prisms and the like. A crucial feature of the angled-dual-illumination-axis focusing means is that it provides first and second illumination axes that intersect optimally at an angle xcex8. The scanning means typically comprises an element selected from the group consisting of mirrors, reflectors, acousto-optic deflectors, electro-optic deflectors, mechanical scanning mechanisms, and Micro-Electro-Mechanical-Systems (MEMS) scanning micro-mirrors. A key feature is that the scanning means is in the form of a single element, as opposed to two or more separate scanning elements in many prior art confocal scanning systems. A preferred choice for the scanning means is a flat pivoting mirror, particularly a silicon micro-machined scanning mirror for its compact and light-weight construction. Moreover, the first and second optical fibers are preferably single-mode fibers, for the ends of single-mode fibers provide nearly point-like light sources and detectors.
A unique feature of the angled-dual-illumination-axis confocal scanning head of the present invention is that the scanning means is placed between the angled-dual-illumination-axis focusing means and the object to be examined. This enables the best on-axis illumination and observation point-spread functions to be utilized throughout the entire angular range of an arc-line scan, thereby providing greater resolution over a larger transverse field of view, while maintaining diffraction-limited (or relatively aberration-free) performance. Such an arrangement is made possible by taking advantage of the longer working distance rendered by using relatively lower NA focusing elements or lenses in the angled-dual-illumination-axis focusing means.
Another important advantage of the angled-dual-illumination-axis arrangement of the present invention is that since each observation beam is positioned at an angle relative to its corresponding illumination beam, scattered (or fluorescent) light along an illumination beam does not easily get passed into its corresponding observation beam, except in the region where the beams overlap. Under certain modes of operation, which are exemplified below, this substantially reduces scattered (or fluorescent) photon noise in the particular observation beam (or beams) being used, thus enhancing the sensitivity and dynamic range of detection. Moreover, by using low NA focusing elements (or lenses) in an angled-dual-illumination-axis confocal scanning system of the present invention, the illumination beams and their corresponding observation beams do not become overlapping until very close to the focus. Such an arrangement further prevents scattered (or fluorescent) light in each illumination beam from directly xe2x80x9cjumpingxe2x80x9d to the corresponding observation beam, thereby further filtering out scattered (or fluorescent) photon noise in the observation beam. Altogether, the angled-dual-illumination-axis confocal system of the present invention has much lower noise and is capable of providing much higher contrast when imaging in a scattering (or fluorescent) medium than the prior art confocal systems employing high NA lenses, rendering it highly suitable for imaging within biological specimens.
A further advantage of the present invention is that the entire angled-dual-illumination-axis confocal scanning head can be mounted on a silicon substrate etched with precision V-grooves where various optical elements are hosted. Such an integrated device offers a high degree of integrity, maneuverability, scalability, and versatility, while maintaining a workable working distance and a large field of view. In particular, a micro-optic version of an integrated, angled-dual-axis confocal scanning head of the present invention can be very useful in biological and medical imaging applications, e.g., endoscopes and hand-held optical biopsy instruments.
As such, the angled-dual-illumination-axis confocal scanning microscope of the present invention is capable of providing an assortment of reflectance and fluorescence images. For instance, a first wavelength-selective-beam-splitting means can be coupled to the first observation beam, diverting a portion of the first observation beam to a first optical detector. The first wavelength-selective-beam-splitting means can be configured to preferentially permit only the reflected light (characterized by a particular wavelength and bandwidth of light) carried by the first observation beam to pass through, thereby providing a first reflectance image signal. A second wavelength-selective-beam-splitting means can be further coupled to the first observation beam, diverting an additional portion of the first observation beam to a second optical detector. The second wavelength-selective-beam-splitting means may be designed to preferentially permit only the particular wavelength and bandwidth of light corresponding to two-photon fluorescence light carried by the first observation beam to pass through, thereby providing a two-photon fluorescence image signal. Likewise, a third wavelength-selective-beam-splitting means can be coupled to the second observation beam, diverting a portion of the second observation beam to a third optical detector. The third wavelength-selective-beam-splitting means can be configured to preferentially permit only the reflected light (characterized by a particular wavelength and bandwidth of light) carried by the second observation beam to pass through, thereby providing a second reflectance image signal. And a fourth wavelength-selective-beam-splitting means may be further coupled to the second observation beam, providing an additional revenue for detecting the two-photon fluorescence light carried by the second observation beam, and so on. All in all, a cascade of the wavelength-selective-beam-splitter means can be optically coupled to either of the first and second observation beams, enabling various components of each of the observation beams to be extracted and detected. Moreover, a superposition of reflectance and two-photon fluorescence images thus obtained would be highly desirable, for it provides complementary information about the morphology and functionality of a biological sample. It should be noted that it is possible to operate the present invention in a number of ways that would provide different combinations of reflectance and fluorescence (single-photon, two-photon, or multiple-photon) images, depending upon the instrument design and the types of light sources/wavelengths used. It is preferable to design the instrument in a way that maximizes the resolution of the images thus produced and that also minimizes the scattered and/or fluorescent photon noise in the image signal. This can be best accomplished by the following seven design rules, which insure that reflected or fluorescence light generated by each illumination beam is optimally collected only by its corresponding (angularly overlapping) observation beam:
1) In the case where the first observation beam is being used to collect reflectance image information characterized by a first wavelength, the second illumination beam should not include light with the first wavelength, and the first illumination beam must provide light with the first wavelength.
2) In the case where the first observation beam is being used to collect single-photon fluorescence image information characterized by a third wavelength when the object is excited by light of a second wavelength, the second illumination beam should not include single-photon excitation light with the second wavelength, and the first illumination beam should provide single-photon excitation light with the second wavelength.
3) In the case where the first observation beam is being used to collect one-color two-photon (1C2P) fluorescence image information characterized by a fifth wavelength when the object is excited by light of a fourth wavelength, the second illumination beam should not include 1C2P excitation light with the fourth wavelength, and the first illumination beam should provide 1C2P excitation light with the fourth wavelength.
4) In the case where either of the first and second observation beams, or both of the observation beams, are being used to collect two-color two-photon (2C2P) fluorescence image information characterized by an eighth wavelength when the object is excited by light that requires both of sixth and seventh wavelengths, the first and second illumination beams should each provide light with only one of the two required wavelengths, such that 2C2P excitation light is provided only in the region where the two illumination beams overlap both spatially and temporally.
5) In the case where the second observation beam is being used to collect reflectance image information characterized by a ninth wavelength, the first illumination beam should not include light with the ninth wavelength, and the second illumination beam must provide light with the ninth wavelength.
6) In the case where the second observation beam is being used to collect single-photon fluorescence image information characterized by an eleventh wavelength when the object is excited by light of a tenth wavelength, the first illumination beam should not include single-photon excitation light with the tenth wavelength, and the second illumination beam should provide single-photon excitation light with the tenth wavelength.
7) In the case where the second observation beam is being used to collect one-color two-photon (1C2P) fluorescence image information characterized by a thirteenth wavelength when the object is excited by light of a twelfth wavelength, the first illumination beam should not include 1C2P excitation light with the twelfth wavelength, and the second illumination beam should provide 1C2P excitation light with the twelfth wavelength.
The present invention further provides an angled-dual-illumination-axis confocal scanning system, comprising an angled-dual-illumination-axis confocal scanning microscope of the present invention, first and second light sources, and first and second optical detectors. The first light source is optically coupled to a second end of the first optical fiber of the angled-dual-illumination-axis confocal scanning microscope by way of a first wavelength-selective-beam-splitting element, providing the first illumination beam. The first wavelength-selective-beam-splitting element additionally diverts a portion of the second observation beam delivered by the first optical fiber to the first optical detector. Likewise, the second light source is optically coupled to a second end of the second optical fiber of the angled-dual-illumination-axis confocal scanning microscope by way of a second wavelength-selective-beam-splitting element, providing the second illumination beam. The second wavelength selective beam-splitting element additionally diverts a portion of the first observation beam delivered by the second optical fiber to the second optical detector. By selecting appropriate first and second wavelength-selective-beam-splitting elements, various spectral components of the first and second observation beams can be extracted and detected according to the aforementioned design rules
In the aforementioned angled-dual-illumination-axis confocal scanning system of the present invention, either of the first and second wavelength-selective-beam-splitting elements can be a dichroic beam-splitter, a dichroic filter, a bandpass filter, a spectral filter, or a wavelength division multiplexer (WDM). Each of the first and second light sources can be a continuous wave (CW) or a pulsed light source, such as a fiber laser, a diode pumped solid state laser, a semiconductor laser, or other suitable fiber-coupled light source known in the art. The optical detector can be a PIN diode, an avalanche photo diode (APD), or a photomultiplier tube. Such an angled-dual-illumination-axis confocal scanning system provides a simple and versatile imaging tool with high resolution and fast scanning capability.
In addition to detecting first and second observation beams, the angled-dual-illumination-axis confocal scanning system of the present invention is equipped to collect a third observation beam comprising predominantly fluorescence light, and directs the third observation beam to an input end of a third optical fiber, as described above. An output end of the third optical fiber can be coupled to a third optical detector, hence providing additional revenue for detecting single-photon or two-photon fluorescence light emanated from the object.
All in all, the angled-dual-illumination-axis confocal scanning microscope of the present invention has advantages of higher resolution, faster scanning, higher sensitivity and larger dynamic range of detection, a larger field of view and a longer working distance, a compact and integrated construction, and great versatility in combining contemporary reflectance and fluorescence imaging.
Moreover, by integrating the angled-dual-illumination-axis confocal scanning microscope of the present invention with fiber-optic components and fiber-coupled laser sources, the angled-dual-illumination-axis confocal scanning systems of the present invention provide a diverse assembly of fiber-based, high resolution and fast scanning systems that can be adapted in a variety of applications, such as in biological and medical imaging, and industrial applications.
The novel features of this invention, as well as the invention itself, will be best understood from the following drawings and detailed description.